Supplementary Material for: Clinical significance of miR-28-5p in ischemic stroke and its neuroprotective effect after ischemia-reperfusion injury

Introduction: Ischemic stroke (IS) remains a leading cause of global morbidity and mortality, necessitating novel therapeutic targets. MicroRNAs (miRNAs) show promise in modulating post-stroke pathology, yet the role of miR-28-5p in IS remains unexplored. This study aimed to investigate miR-28-5p expression in IS and its clinical and mechanistic significance. Methods: Serum miR-28-5p levels were quantified via qRT-PCR in 129 IS patients and 97 healthy controls. Kaplan-Meier (K-M) analysis and multivariate COX regression analysis evaluated the prognostic value of miR-28-5p in IS. In vitro, dual-luciferase assays validated the interaction between miR-28-5p and WNK3. Oxygen-glucose deprivation/reoxygenation (OGD/R)-treated HMC3 microglia were used to assess miR-28-5p/WNK3 axis in microglial polarization, oxidative stress, and inflammation. Results: Serum miR-28-5p was significantly downregulated in IS patients, associated with elevated TG, LDL-C, reduced HDL-C levels, and higher NIHSS scores. K-M analysis revealed a lower progression-free survival (PFS) rate in IS patients with low miR-28-5p expression, and COX regression analysis confirmed miR-28-5p as an independent prognostic factor. In mechanism miR-28-5p directly targeted WNK3. Overexpression of miR-28-5p suppressed OGD/R-induced M1 polarization, oxidative stress (reduced MDA, elevated SOD), and inflammatory cytokines (decreased TNF-α, IL-6, IL-1β, and increased IL-10), whereas WNK3 overexpression reversed these effects. Conclusion: MiR-28-5p served as a diagnostic biomarker and independent prognostic factor in IS. Its neuroprotection involved suppressing WNK3-mediated microglial M1 polarization, oxidative stress, and inflammation, highlighting its potential as a therapeutic target for IS.